1. Field of the Invention
The present invention relates to a pharmaceutical composition for the prevention or treatment of diseases associated with abnormal cell proliferation, comprising a cytosolic Hsp60 inhibitor, and a method and kit for screening a therapeutic agent for diseases associated with abnormal cell proliferation using cytosolic Hsp60.
2. Description of the Related Art
Mammalian cells express a number of survival genes that play the roles in inhibiting caspase activation, removing harmful oxygen radicals, defending mitochondrial function, and checking cell cycle. Among the transcription factors responsible for the induction of the survival genes, nuclear factor-Kb(NF-κB) is a key element that orchestrates the complex cell survival response. In particular, the NF-κB-dependent survival genes include antiapoptotic genes, such as c-IAPs and c-FLIP, and mitochondrial safeguard genes, such as manganese-superoxide dismutase(MnSOD) and Bcl-2 family members.
A central kinase in the NF-κB activation pathway is the inhibitor of κB kinase (IκB kinase or IKK) that phosphorylates the IκB protein in amino-terminal serine residues, leading to its ubiquitinylation and proteosomal degradation and to the consequent liberation of NF-κB proteins [Karin M et al., Annu Rev Immunol 18:621-663 (2000)]. The extracellular stimuli to activate NF-κB pathway converge to IKK [Hayden M S et al., Genes Dev 18: 2195-2224 (2004)]. Therefore, numerous efforts have been made to delineate the regulation of IKK activation.
Specifically, that the IKK activation is regulated by dependent upon phosphorylation has already been known. The phosphorylation of two serine residues (Ser177/Ser181 in human IKKβ) in activated T-loop is essential for the activation thereof, while the autophosphorylation of C-terminal serine cluster turns off the activation. Many kinases have been implicated as being involved in the activation phosphorylation: NF-κB inducing kinase (NIK), mitogen-activated protein kinase/ERK kinase kinases 1 (MEKK1), MEKK2/3, hematopoietic progenitor kinase-1 (HPK1), mixed-lineage kinase 3(MLK 3), and TGF-β activated kinase 1(TAK1). However, except for in the case of TAK1, it is unclear how the upstream kinases activate the IKK complex.
Further, the ubiquitin-dependent regulation of IKK activation has been studied for many years. Recently, the regulatory subunit IKKγ (or NEMO) of IKK complex has been shown to be ubiquitinated, as well as to recognize Lys63-linked polyubiquination chain on RIP 1 (receptor-interaction protein 1).
Further, the regulation of IKK activation via the interacting proteins has already been known. The best examples are heat shock proteins. For example, Cdc37 and Hsp90 have been reported to act as additional components of the IKK complex that stabilize the complex [Chen G et al., Mol Cell 9:401-410 (2002)]. Hsp27 has been shown to interact with IKKβ in a TNF-α-dependent manner [Park K J et al., J. Biol. Chem. 278:35272-35278 (2003)]. Hsp70 also interacts with IKKγ but interferes with the IKK activation [Ran R et al. Genes Dev 18:1466-1481 (2004)]. In addition, the association between protein phosphatase 2Cβ (PP2Cβ) and the IKK complex has already been demonstrated, and ELKs have also been identified as a new regulatory subunit of IKK complex that mediates the recruitment of IκB to the complex. However, there is no indication of a mitochondrial protein involved in the IKK/NF-κB activation.
Meanwhile, the molecular chaperones Heat Shock Protein 90 (Hsp90), Rsp60, and Heat Shock 70 kDa Protein 9 (HSPA9/mortalin) are found at increased levels in mitochondria of tumor cells, and “mitochondrial-targeted” chaperone inhibitors are known to be used for the treatment of disorders associated with unwanted cell proliferation [WO09/036,092].
The present inventors have made many efforts to understand the mechanism of NF-κB activation. As a result, they identified that Ksp60, which functions as a Heat Shock Protein in the mitochondria, directly interacts with IKKα/β in the cytoplasm and then promotes the phosphorylation-dependent activation of the kinase, and subsequently increased transcriptional activity of NF-κB induced survival genes to reduce the intracellular reactive oxygen species (ROS) level, leading to an increase in cell survival against apoptosis-inducing stress. Accordingly, they found that abnormal cell proliferation-associated diseases can be treated by inhibiting cytosolic Hsp60 expression and/or activation, thereby completing the present invention.